Utilizing Targeted Gene Therapy With Nano Particles Binding Alpha v Beta 3 for Imaging and Treating...

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Utilizing Targeted Gene Therapy with NanoparticlesBinding Alpha v Beta 3 for Imaging and TreatingChoroidal Neovascularization

Hani Salehi-Had1., Mi In Roh1., Andrea Giani1, Toshio Hisatomi1, Shintaro Nakao1, Ivana K. Kim1,

Evangelos S. Gragoudas1

, Demetrios Vavvas1

, Samira Guccione2

*, Joan W. Miller1

1 Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States of

America, 2 Radiological Sciences Laboratory, Lucas Center, Stanford University, Palo Alto, California, United States of America

Abstract

Purpose: The integrin avb3 is differentially expressed on neovascular endothelial cells. We investigated whether a novelintravenously injectable avb3 integrin-ligand coupled nanoparticle (NP) can target choroidal neovascular membranes (CNV)for imaging and targeted gene therapy.

Methods: CNV lesions were induced in rats using laser photocoagulation. The utility of NP for in vivo imaging and genedelivery was evaluated by coupling the NP with a green fluorescing protein plasmid (NP-GFPg). Rhodamine labeling (Rd-NP)was used to localize NP in choroidal flatmounts. Rd-NP-GFPg particles were injected intravenously on weeks 1, 2, or 3. In thetreatment arm, rats received NP containing a dominant negative Raf mutant gene (NP-ATPm-Raf) on days 1, 3, and 5. The

change in CNV size and leakage, and TUNEL positive cells were quantified.

Results: GFP plasmid expression was seen in vivo up to 3 days after injection of Rd-NP-GFPg. Choroidal flatmountsconfirmed the localization of the NP and the expression of GFP plasmid in the CNV. Treating the CNV with NP-ATPm-Rafdecreased the CNV size by 42% (P,0.001). OCT analysis revealed that the reduction of CNV size started on day 5 andreached statistical significance by day 7. Fluorescein angiography grading showed significantly less leakage in the treatedCNV (P,0.001). There were significantly more apoptotic (TUNEL-positive) nuclei in the treated CNV.

Conclusion: Systemic administration of avb3 targeted NP can be used to label the abnormal blood vessels of CNV forimaging. Targeted gene delivery with NP-ATPm-Raf leads to a reduction in size and leakage of the CNV by induction ofapoptosis in the CNV.

Citation: Salehi-Had H, Roh MI, Giani A, Hisatomi T, Nakao S, et al. (2011) Utilizing Targeted Gene Therapy with Nanoparticles Binding Alpha v Beta 3 for Imagingand Treating Choroidal Neovascularization. PLoS ONE 6(4): e18864. doi:10.1371/journal.pone.0018864

Editor: Sotirios Koutsopoulos, Massachusetts Institute of Technology, United States of America

Received December 15, 2010; Accepted March 21, 2011; Published April 29, 2011Copyright: 2011 Salehi-Had et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by: Research to Prevent Blindness (NY) Unrestricted Grant to the Harvard Department of Ophthalmology and NeovascularResearch Fund; AMD Center of Excellence. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript.

Competing Interests: The authors have no financial interests to disclose. Samira Guccione is a named inventor on the patent application for the therapy used inthis paper. There is no commercialization of the application. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials.

* E-mail: guccione@stanford.edu

. These authors contributed equally to this work.

Introduction

Age-related macular degeneration (AMD) is the leading cause of

blindness in developed countries for people over the age of 50[13]. The neovascular or wet form of the disease, characterized

by the development of choroidal neovascular membranes (CNV) is

the main cause of visual impairment in macular degeneration

[35]. With the advent of new treatment options such as

photodynamic therapy, and especially intravitreal antiangiogenic

pharmacotherapy, the visual prognosis of patients with CNV has

improved significantly [69]. However, the current standard-of-

care therapies require monthly intravitreal injections by a retina

specialist due to their short half-life in the vitreous [10,11]. Aside

from the logistic difficulties and the patients discomfort, it also

puts the patient at risk for cataract formation, endophthalmitis,

vitreous hemorrhage, and retinal detachment. Thus, there is a

great need for alternative means of delivering antineovascular

therapy to the retina.

Recently, there has been substantial progress in the develop-ment of nanoparticles with an integrin-targeted delivery system

[1215]. During vascular remodeling and angiogenesis, several

integrins are expressed on the endothelial cells to potentiate cell

invasion and proliferation [16,17]. Among them, integrin avb3 is

expressed on many cell types but its expression level in normal

tissue is generally low [18,19]. It is preferentially expressed on

angiogenic blood vessels, mediating survival signal and facilitating

vascular cell proliferation [20,21]. Previous reports show that

integrin avb3 is involved in ocular angiogenesis [22,23]. In vivoexperiments have shown antibodies blocking or immunoconjugate

drug therapy targeting integrin avb3 inhibit neovascularizaion

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[17,2326]. In addition, integrin avb3 potentiates the internaliza-

tion of various viruses [27,28], making it a potential target for drug

delivery via liposome based nanoparticles.

Previously we have shown that systemic injection of a cationic

nanoparticle coupled to an integrin avb3-targeting ligand (NP) can

deliver a suicide gene to the tumor neovasculature in rats, causing

apoptosis and significant tumor regression [12]. Here we evaluated

and were able to demonstrate that NP can target choroidal

neovascular membranes (CNV) in rats for imaging and targetedgene therapy using a plasmid DNA encoding ATPm-Raf, a

dominant-negative mutant form of Raf kinase [29].

Materials and Methods

Animals and Ethics StatementAll experiments were conducted in accordance with the recom-

mendations in the Guide for the Care and Use of Laboratory Animals

of the National Institutes of Health.and the guidelines established by

the Animal Care Committee (ACC) of the Massachusetts Eye and Ear

Infirmary. The protocol was approved by the ACC (protocol number

07-10-012). A total of 106 Brown-Norway male rats weighing 175

225 grams were obtained from Charles River Laboratories

(Wilmington, MA) and used for the experiments.

Characteristics and preparation of NanoparticlesDetailed description of the NPs and their synthesis has been

published previously [12]. All custom-made lipids and genes were

GLP manufactured. Briefly, purified lipid components were

dissolved in organic solvents (CHCl3 and CH3OH in a ratio 1:1).

The CHCl3 and CH3OH were evaporated and dried in rotavap for

24 hours. Distilled and deionized water was added to yield a

heterogeneous solution of 30 mM in total lipid concentration. The

lipid/water mixture was then sonicated with a probe-tip sonicator

for at least one hour. Throughout sonication, the pH of the solution

was maintained between 7.0 and 7.5 with 0.01N NaOH solution,

and the temperature was maintained above the gel-liquid crystal

phase transition point (Tm). The liposome solution was transferred

to a petri dish resting on a bed of wet ice, cooled to 0uC, and

irradiated at 254 nm for at least one hour with a hand-held UV

lamp placed 1 cm above the petri dish, yielding NPs. The NPs were

then filtered through a 0.2 mm filter and collected.

Using a Brookhaven dynamic light scattering system (DLS), the

size (diameter), distribution, and zeta potential of NPs weredetermined to be 45.3+2.4 nm and +35mv respectively, averaged

for 17 cycles of NP synthesis.

The rats received intravenous treatments at a dose 1 mg/kg of

NP and 1 mg/kg of plasmid DNA containing the Raf mutant gene

(ATPm-Raf). Total volume of injection was 350 ml.

Induction of Choroidal Neovascular MembranesAnimals were anesthetized with an intraperitoneal injection of

0.2 to 0.3 mL of a 1:1 mixture of 100 mg/mL ketamine and

20 mg/mL xylazine. Pupils were dilated with 5.0% phenylephrine

and 0.8% tropicamide. CNV was induced in the eyes of rats with a

532-nm laser (Oculight GLx; Iridex, Mountain View, CA), as

previously described [3032]. Four to eight laser spots (180 mW,

100 mm, 100 ms) were placed in each eye of the rat with a slit

lamp delivery system and a cover slip serving as a contact lens. Ifsignificant hemorrhage occurred, the eye was excluded.

Evaluation of Specific Targeting of CNV Using In VivoImaging and Choroidal Flatmounts

NP carrying a green fluorescing protein (GFP) plasmid (NP-

GFPg) was used to evaluate the ability of the particles to deliver a

gene to the neovascular endothelial cells. Rhodamine labeling of

the NP (Rd-NP) was used to localize the particles in choroidal

flatmounts. Rats were divided into 3 groups of 6 animals and

evaluated 1, 2, or 3 weeks after creation of CNV. The formation of

Figure 1. Bioimaging with NP-angiography showing GFP expression using the Topcon camera with fluorescein angiography filtersettings. Late phase FAs (A and D) show the CNV lesions prior to injection of NP. Autofluorescent images taken prior to injection of NP revealminimal background fluorescence of the CNV lesions (B and E). Injection of targeted NP carrying a GFP plasmid (NP-GFPg) causes increasedfluorescence of the CNV lesions from GFP expression (C) whereas non-targeted NP carrying a GFP plasmid (ntNP-GFPg) does not cause any increase inthe intensity of fluorescence of the CNV over background autofluorescence (F).doi:10.1371/journal.pone.0018864.g001

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CNV was confirmed by fluorescein angiography (FA) using a

digital fundus camera (TCR50IA; Topcon, Paramus, NJ)

following intraperitoneal injection of 0.2 mL of 2% fluorescein

sodium. The rats where then injected with Rd-NP-GFPg particles.

The expression of GFP was evaluated with in vivo imaging using

the same camera and FA filter settings 24, 48, and 72 hours after

injection of particles. The rats were then euthanized and choroidal

flat mounts were performed at the above time points. RPE-

choroid-sclera complex was flatmounted (Vector Laboratories,Burlingame, CA) and coverslipped. Pictures of the choroidal

flatmounts were taken by a confocal microscope (Leica Micro-

systems, Wetzler, Germany). Negative controls were evaluated

under identical conditions without injection of NP or following

injection of non-targeted rhodamine labeled liposome particles

carrying GFP-plasmid.

Treatment of CNV with Targeted Gene DeliveryAfter creation of laser-induced CNV, animals were divided in to

7 groups; groups AC were treatment groups and groups DG

were controls. Group A (n = 6 received one intravenous injection

of anb3 targeted-NP containing ATPm-Raf (NP-ATPm-Raf) on

days 1, 3, and 5 after CNV creation; group B (n = 6) received one

intravenous injection of NP-ATPm-Raf on days 3, 5, and 7; group

C (n = 3) received one intravenous injection of NP-ATPm-Raf on

days 7, 9, and 11; group D (n = 6) did not receive any treatment;

group E (n = 3) received one intravenous injection of non-targeted

NP containing ATPm-Raf (ntNP-ATPm-Raf) on days 1, 3, and 5;

group F (n= 3) received one intravenous injection of anb3

targeted-NP without ATPm-Raf (NP) on days 1,3, and 5; and

group G (n = 3) received one intravenous injection of ATPm-Raf

gene without NP on days 1, 3, and 5.

Evaluation of CNV Size and LeakageWe used FA and OCT to monitor CNV development and

changes in vivo and choroidal flatmounts to study the size of the

lesions ex vivo. FA was performed as detailed above, 1 and 2 weeks

after CNV creation in all treated and control animals. A choroidal

neovascular membrane was defined as fully regressed aftertreatment if there was no leakage in the area of treated membrane

[30,31]. The angiograms were graded by two masked readers

using a pre-established grading scheme [33,34]. Briefly, the

description of each grade follows: 0, faint hyperfluorescence or

mottled fluorescence without leakage; 1, hyperfluorescent lesion

without progressive increase in size or intensity; 2A, hyperfluor-

escence increasing in intensity but not in size; 2B, hyperfluores-

cence increasing in intensity and in size.

Two weeks after CNV creation, the size of the CNV lesions was

measured in choroidal flatmounts using the methods reported

previously after perfusion of 5 mg/mL fluorescein labeled dextran

[31,35]. A computer program (OpenLab; Improvision, Boston,

MA) was used by two masked investigators to measure the

hyperfluorescent areas corresponding to the CNV lesions.

Optical Coherence Tomography Measurement of CNVSize

Six rats treated with intravenous NP-ATPm-Raf on days 1,3 and

5 and 6 control rats without treatment were used for evaluation of

CNV size in vivo using optical coherence tomography (SDOCT,

Bioptigen, Durham, NC) on days 3, 5, and 7 after CNV creation.

A volume analysis was performed, using 100 horizontal raster,

consecutive B-scan lines, each one composed of 1200 A-scans. The

volume size was 2.162.1 cm. To evaluate the cross-sectional size

of each lesion in OCT images, the sections passing through the

center of the CNV were chosen. The center of the lesion was

defined as the midline passing through the area of RPE-Bruchs

membrane rupture. In order to consistently identify this point, we

used the en-face fundus reconstruction tool provided with the

Bioptigen SD-OCT system. For each time point, the same spot

was used to evaluate the size of the CNV. CNV was outlined from

the inner border of the retinal pigment epithelial layer to the top of

the lesion and the size was measured using Image J software

(http://rsbweb.nih.gov/ij/, last access January 7th

2009).

Histopathology of CNV LesionsOn day 3,5, 7 and 14 after CNV creation, eyes were enucleated

and fixed in 4% paraformaldehyde in phosphate-buffered saline

Figure 2. Choroidal flatmounts showing accumulation ofrhodamine labeled NP and expression of GFP plasmid in theCNV. The CNV lesions are delineated by arrowheads in bright fieldimages with false blue color (A and E). FITC-filtered images highlight theGFP expression one day after systemic injection of Rd-NP-GFPg (B)whereas non-targeted NP (Rd-ntNP-GFPg) does not induce GFPexpression in CNV (F). Cy3-filtered images highlight that rhodamine-labeled NP (Rd-NP-GFPg) accumulates in the CNV (C), while rhodamine-labeled non-targeted NP (Rd-ntNP-GFPg) does not (G). Some particlescan be visualized circulating in the choroidal vessels. Overlay of imagesAC is presented in panel D and overlay of EG is shown in H.doi:10.1371/journal.pone.0018864.g002

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(PBS) for 1 hour and cryoprotected. Serial sections of the eyes

were cut at 10 mm thickness on a cryostat (CM1850; Leica,

Heidelberger, Nussloch, Germany) at 220 C, and prepared for

staining. Terminal dUTP Nick-End Labeling (TUNEL) assay was

performed according to the manufacturers protocol (ApoTag

Fluorescein in situ Apoptosis Detection Kit; Chemicon, Temecula,

CA) as previously reported [31,36]. CD31 (1:100, Serotec,

Oxford, UK) antibody was used for visualizing endothelial cells

and a mouse monoclonal antibody for ED1, the rat homologue ofhuman CD68 (1:100, Millipore, Billerica, MA) was used for

staining macrophages. Sections were then stained with DAPI

(1:1000, Invitrogen Ltd, Carlsbad, CA, USA) for nuclear staining

and mounted with Vecta shield mounting media (Vector

Laboratories, Burlingame, CA). Photographs of the CNV were

taken with upright fluorescent microscope (DM RXA; Leica,

Solms, Germany) and the number of TUNEL positive and ED 1

positive cells were counted.

Statistical Analysis

All values are presented as mean6

SE. Paired groups werecompared using the Wilcoxon t-test. For three groups, data were

compared by Kruskal-Wallis test and for two group comparisons,

Figure 3. Late phase fluorescein angiography (FA) and choroidal flatmounts (x10) two weeks after laser photocoagulation.Representative lesions are from the control group (AD) and the NP-ATPm-Raf treated group (E and F). Group (A) received no treatment; (B) receivedintravenous injection of non-targeted NP containing ATPm-Raf on days 1, 3, and 5 after laser CNV creation; (C) received intravenous injection of a

nb3

targeted-NP without ATPm-Raf gene on days 1,3, and 5; (D) received injection of ATPm-Raf gene without NP on days 1, 3, and 5; (E) received injectionofa

nb3 targeted-NP containing ATPm-Raf (NP-ATPm-Raf) on days 1, 3, and 5; and (F) received injection of NP-ATPm-Raf on days 3, 5, and 7. NP-ATPm-Raf

treated groups (E and F) had significantly lower grade CNV lesions on FA grading and smaller CNV size compared to the control group (AD). Nostatistically significant difference in size was noted between the control groups AD. Quantification of the CNV size on choroidal flat mounts is shownin (G). *P,0.01. Data are expressed as the mean 6 SE.doi:10.1371/journal.pone.0018864.g003

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Mann-Whitney U test was used (SPSS statistics 17.0, SPSS Inc.,

Chicago, IL, USA). A P value of less than 0.05 was considered

statistically significant.

Results

In Vivo imaging Reveals GFP-Plasmid Expression by CNVFormation of CNV was confirmed by FA prior to injection of

NP. One day after injection of rhodamine-labeledavb

3 targetednanoparticle carrying a GFP plasmid (Rd-NP-GFPg), digital

fundus photography with FA filter settings revealed hyperfluores-

cence of the CNV lesions, confirming localization and adhesion of

the NP and GFP expression. This hyperfluorescence was sustained

through day 3 (data not shown) and was seen in 1, 2, and 3-week-

old CNV lesions examined (Figure 1AC). There was no notable

hyperfluorescence noted above background fluorescence level in

the control groups (Figure 1DF). There was no evidence of

increased fluorescence in the normal retinal or choroidal

vasculature.

Rhodamine-Labeled avb3 Targeted NanoparticleAccumulate in the CNV and Induce GFP Expression

The delivery of rhodamine dye and the expression of GFP

plasmid in the CNV were confirmed by performing confocal

microscopy on choroidal flat mounts (Figure 2). Choroidal

flatmounts performed after in vivo imaging revealed accumulation

of rhodamine labeled nanoparticles in the CNV lesion (Figure 2C).

There was an overlap of GFP expression and the rhodamine

accumulation in the CNV (Figure 2D). No notable difference was

seen in the pattern of NP accumulation or intensity of GFP

expression over the time course examined. There was no evidence

of increased fluorescence in the normal choroidal vasculature and

no evidence of rhodamine accumulation or GFP expression in the

control animal injected with non-targeted rhodamine labeled

nanoparticles carrying a GFP plasmid (Rd-ntNP-GFPg;

Figure 2EH).

anb3 Targeted-NP Containing ATPm-Raf Reduces the Sizeof CNV

Treatment of CNV with intravenous injection ofanb3 targeted-NP containing ATPm-Raf (NP-ATPm-Raf) on days 1, 3, and 5resulted in a 42.0% reduction in the CNV size on choroidal

flatmount compared with control CNVs with no treatment (mean

size, 53538.7 mm2 vs. 31029.3 mm2, p,0.001) (Figure 3A and E).

Treatment with 3 doses of NP-ATPm-Raf on days 3, 5 and 7 also

showed a 24.6% decrease in the CNV size (Figure 3F). Treatment

on days 7, 9, and 11 did not lead to a significant reduction of CNV

size (data not shown). Three additional control groups (n = 3 each)

were given intravenous injection of non-targeted-NP containing

ATPm-Raf (ntNP- ATPm-Raf), naked ATPm-Raf, and anb3targeted-NP with no ATPm-Raf (NP) on days 1,3, and 5 afterCNV formation. There was no difference in the CNV size

between any of the 4 control groups, including the no treatmentgroup (Kruskal- Wallis test, p = 0.168) (Figure 3AD).

To track the reduction of CNV size in vivo after treatment with

NP-ATPm-Raf, we used OCT imaging. We were able to delineatethe CNV as a discrete subretinal hyperreflective material starting

on day 5 (Figure 4B and C). While the difference in the cross-

sectional CNV size on day 5 between treated and control CNV

had not reached statistical significance, (Mann- Whitney U test,

p = 0.066), significantly decreased size was noted on day 7 in the

treated group compared to the control group (Mann- Whitney U

test, p = 0.001) (Figure 4A).

Treatment with NP-ATPm-Raf Leads to a Reduction in Sizeand Leakage of CNV on FA

Quantitative assessment of CNV leakage by FA performed on

day 14 was carried out by two masked graders. Pathologic leakage

from the CNV could be noted in the late phase (68 minutes) in

the untreated group with large and diffuse area of leakage

(Figure 3AD). The treated group showed less leakage with

smaller lesions (Figure 3E and F). FA grading revealed that

70%,83.8% of the CNV in the different control groups showed

grade 2B leakage, while 40.3% of the CNV in the treated group

had grade 2B leakage (Table 1; Pearson Chi- square test,

p,0.001). There was no difference in the distribution of grading

amongst the 4 different control groups (Pearson Chi- square test,

p = 0.679). Also, no statistically significant difference was found in

the distribution of leakage grading between the groups treated ondays 1, 3, and 5 or days 3, 5, and 7 (Pearson Chi- square test,

p = 0.152).

Reduced Endothelial Cell Count and Increased Apoptosisin CNV Treated with NP-ATPm-Raf

CD31-positive endothelial cells were detected in the subretinal

space starting on day 3 after laser injury (figure 5D). The cells

increased and focalized to a distinct subretinal membrane

representing the CNV by day 7. In the treated CNV, there were

Figure 4. In vivo evaluation of CNV utilizing SD-OCT. Quantification of CNV size using SD-OCT (A) reveals a decrease in CNV size, reachingstatistical significance on day 7 (Mann- Whitney U test, p = 0.001) in the NP-ATPm-Raf treated group compared to the control group. A hyper-reflectivesubretinal lesion is seen as delineated by the red dotted line (B). This lesion corresponds to the hyporeflective area on fundus reconstruction (reddotted circle, C). *P,0.01. Data are expressed as the mean 6 SE.doi:10.1371/journal.pone.0018864.g004

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fewer CD 31-postive cells and the CNV appeared more compact

with distinct borders and fibrous formation by day 7 (Figure 5D).

We investigated signs of apoptosis with TUNEL staining. In the

treated group, significantly more TUNEL-positive nuclei were

observed in the CNV starting on day 3 (Figure 5A and B). This

trend continued through day 7 (Mann Whitney U test, p,0.01 for

day 3 and 5, p = 0.01 for day 7, Figure 6B). The reduction of the

CNV size as measured in histological sections reached statistical

significance on day 7 (Mann Whitney U test, P = 0.001, Figure 6C).

Macrophage infiltration with anb3 Targeted-NPContaining ATPm-Raf

ED 1 positive cells (a marker for macrophages, equivalent tohuman CD 68) were concentrated within the subretinal space at

the laser injury site on day 3 (Figure 6D). No ED 1-positive cells

were observed in the undamaged choroid. No difference was

noted in the number of ED 1-positive cells infiltrated into the

CNV between the treated group and the untreated group on day

3. However, on day 5 and 7 statistically more ED 1-positive cells

were seen in the treatment group (Mann Whitney U test, P,0.01

respectively; Figure 6D).

Discussion

Specific targeting and delivery of medication for the treatment

of CNV remains challenging [37]. In this study we were able to

target experimental CNV after systemic injection of a cationic

Table 1. Fluorescein angiography (FA) grading of CNVlesions.

G ra de 1 G ra de 2 A G ra de 2 B Tot al

Control 5(6.2%) 16(19.8%) 60(74.1%) 81

ntNP-ATPm-Raf 2(5%) 10(25%) 28(70%) 40

ATPm-Raf 3(7.1%) 7(16.7%) 32(76.2%) 42NP 0(0%) 6(16.2%) 31(83.8%) 37

NP-ATPm-Raf on D1, 3, 5 10(13.9%) 33(45.8%) 59(40.3%) 72

NP-ATPm-Raf o n D3 , 5, 7 1 (2. 8%) 1 6(44 .4%) 1 9(5 2. 8%) 36

Significantly higher percentage of control CNV lesions were Grade 2B comparedto the treated CNV (Pearson Chi-square test P,0.001). There were no significantdifferences between the different treatment groups (Pearson Chi-square testP = 0.679).doi:10.1371/journal.pone.0018864.t001

Figure 5. Evaluation of endothelial cell apoptosis with TUNEL staining in frozen sections. Quantification of TUNEL positive cells showedsignificantly more TUNEL(+) cells/lesion (A) and TUNEL (+) cells/mm2 (B) with treatment of NP-ATPm-Raf compared to the control group on day 3 and5 after laser injury. There was a statistically significant reduction of CNV size noted on day 7(C). Double-immunofluorescent staining of frozen sections(x20) obtained at 3, 5 and 7 days after laser photocoagulation for the endothelial cell marker CD31 and TUNEL stain (D). *P,0.01. Data are expressedas the mean 6 SE.doi:10.1371/journal.pone.0018864.g005

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nanoparticle coupled to an integrin avb3-targeting ligand (NP)

and utilize this method for imaging and treatment of CNV in rats.

We first demonstrated the vascular targeting of NP and its

ability to deliver a gene to the neovascular endothelial cells of

CNV in rats using rhodamine labeled NPs coupled with GFP (Rd-

NP-GFPg). GFP expression was seen in vivo using fundus NP-

angiography with FA filter settings and ex vivo in choroidal flatmounts. In vivo imaging revealed increased fluorescence, sustained

for 3 days after NP injection, in 1, 2, or 3 week old CNV (figure 1).

GFP expression co-localized to the area of rhodamine labeled NP

accumulation in the CNV on choroidal flat mounts indicatingGFP expression is correlated with areas of NP adhesion to

neovascular endothelial cells of CNV (figure 2). None of the retinal

or choroidal vasculature showed increased fluorescence from GFP

expression. The specific targeting of NP is due to the selectivity of

its binding ligand for integrin avb3 [12], which has limited cellulardistribution in normal tissue including the eye [23,38]. This

integrin is significantly up regulated during the process of vascular

remodeling and angiogenesis and is present in pathologic

specimens from human eyes with CNV or proliferative diabetic

retinopathy (PDR) [17,22,23,39,40]. With the specific labeling of

CNV demonstrated here, fundus NP-angiography utilizing various

dyes has the potential to be a novel imaging technique for

detecting new CNV or to follow CNV activity independent of

CNV size and leakage. Recently, Takeda and colleagues have

shown early detection of experimental CNV, not visible on FA,

using a similar technique with anti-CCR3 antibody fragments

[41]. The advantage of using NPs over immunoconjugate dyes in

bioimaging and targeted therapy is that they are less likely to incite

an immune reaction.

In the treatment arm of the study, we were able to demonstrate

significant reduction of CNV size and leakage by targeted gene

therapy using NP coupled to ATPm-Raf, a dominant negativeform of Raf kinase (Figure 3, 4). Previously Singh et al treated

experimental CNV using poly-lactide-co-glycolide (PLGA) nano-

particles carrying a VEGF inhibitory gene [42]. Our approach is

different in a number of ways including the type of particles used,

specificity of targeting, and the gene delivered. The particles that

Singh et al used had a negative z-potential as expected for PLGA

nanoparticles, our therapy has a neutral charge and therefore is

not toxic in tissue-cultured cells. Our nanoparticles are smaller

(45.3 nm vs. 270420 nm) and form a stable shell through

covalent bonds that are far more stable in blood circulation than

polymer based (used by Sing et al and hydrolyzed in aqueous

Figure 6. Increased macrophage infiltration at the site of treated CNV. Macrophage infiltration was highest on day 3 with gradual decreaseon days 5 and 7. Significantly higher number of macrophages were observed with the NP-ATPm-Raf treated group compared to the control group on

days 5 and 7 (A and B). There was a statistically significant reduction of CNV size noted on day 7(C). Immunofluorescent staining of representativefrozen sections (x20) obtained at 3, 5, and 7 days after laser photocoagulation for ED 1, a marker for macrophage (D). *P,0.01. Data are expressed asthe mean 6 SE.doi:10.1371/journal.pone.0018864.g006

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environment) or liposome based delivery systems. This stability

may lead to more efficacy and/or more side effects. A separatetoxicity study is planned to answer this question. The targeted

gene, Raf kinase, is an integral member of an intracellular signaltransduction pathway involved in regulation of the cell cycle.

ATPm-Raf is a mutant form of Raf-l that fails to bind ATP andblocks the endothelial cell Raf activity in vitro [12,29]. Raf-1

mutation has been linked to vascular defect and apoptosis during

embryogenesis and gene therapy with ATPm

-Raf causes endothe-lial cell apoptosis and tumor regression in rats [12,43]. Our results

indicate a similar mechanism of CNV regression, through

induction of apoptosis in neovascular endothelial cells, after

targeted gene therapy with NP-ATPm-Raf. There were signifi-cantly higher number of TUNEL positive cells in the treated CNV

as compared to the controls (Figure 5). Moreover, while the

recruitment of macrophages per lesion decreased with time in both

groups, more macrophage infiltration was noted on days 5 and 7

in the treatment group (Figure 6). The initial spike in macrophage

infiltration is likely the result of the inflammatory response to the

laser injury. The increased macrophage recruitment to the treated

CNV closely follows the increased apoptotic activity, suggesting

that the macrophages may be responding to cell death by

apoptosis or necroptosis.

Repeated systemic administration of NP for the treatment ofCNV may lead to side effects such as blood clots in the elderly

patient population. However, Intravenous (I.V.) administration of

our treatment is not repetitious as apposed to the intravitreal

injections of anti-VEGF therapies for example. In addition, for

some patients, I.V. injections are less invasive than intravitreal

injections and do not carry the potential ocular complications.

Furthermore, due to the specificity of the binding site, the dose of

nanoparticles needed is small and the tissue distribution limited,

making systemic side effects less likely. In our study, we did not

attempt the intreavitreal route of delivery as part of the

investigation. Due to the large size of the NP (45.3 nm), it is

unclear if it can distribute through the vitreous and cross the retina

to reach the lumen of the CNV vessels through phagocytosis,

pinocytosis or other mechanisms.

We also explored the timing of imaging and treatment with

targeted NP in our study. Although we were able to target CNV

with NP and capture GFP expression by NP-angiography up to

three weeks after CNV induction, the maximum efficacy of

treatment was achieved when treatment was given on days 1,3 and

5 (42% reduction in CNV size). We saw a modest decrease in the

CNV size (24.6%) when the treatment was given on days 3, 5 and

7 and found no effect with treatment on days 7, 9 and 11. This

data is consistent with the timing of integrin avb3 expression in thelaser induced CNV in rats [44]. Integrin avb3 is expressed up to 4

weeks after laser injury, however its expression levels are

significantly higher in the early stages of CNV formation, peaking

at day 7 [44]. The fact that we were unable to show CNV

regression with treatment starting at later time points may be due

to the concentrations of NP used or the efficacy of transfecting the

CNV endothelial cells with ATPm-Raf. Integrin avb3 expression

has been shown in pathologic specimens from human CNV and

diabetic retinopathy [23,45], however, the timing and duration of

expression of this integrin has not been studied.

In summary, our results provide evidence that systemic

administration of avb3 targeted NP can be used to label the

abnormal blood vessels of CNV for imaging and targeted gene

therapy with ATPm-Raf. These results provide a proof-of-concept

for this emerging technology and encourage further experimen-

tation to discern the integration of NPs into current imaging and

treatment of CNV and retinal neovascularization. Large animal

studies and experiments utilizing NP coupled to various dyes and

chemotherapeutic or anti-neovascular compounds need to be

pursued to further explore the efficacy of this diagnostic and

treatment strategy.

Author Contributions

Conceived and designed the experiments: HS-H SG JWM TH DV.

Performed the experiments: HS-H MIR AG TH SN IKK ESG SG.

Analyzed the data: HS-H MIR AG SG JWM DV. Contributed reagents/

materials/analysis tools: SG JWM DV TH SN. Wrote the paper: HS-H

MIR AG SG JWM DV.

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